Transcription factors represent a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes. One family of transcription factors, CCAAT/Enhancer-binding proteins (C/EBPs), regulates the expression of an extensive panel of genes that control normal tissue development and cellular function, cellular proliferation and functional differentiation. Six members of this family have been identified to date all of which form both homo- and heterodimers with other C/EBP family members as well as with members of the NFkB and Fos/Jun families of transcription factors (Lekstrom-Himes and Xanthopoulos, J. Biol. Chem., 1998, 273, 28545-28548). While all of the members of the C/EBP family have a similar modular protein structure, expression levels and tissue distributions vary widely leading to a diversity of roles (Lekstrom-Himes and Xanthopoulos, J. Biol. Chem., 1998, 273, 28545-28548).
C/EBP beta (also known as C/EBP2, LAP, TCF5, CRP2, NFIL6, IL6DBP, NF-M, AGP/EBP and Apc/EPB) was originally identified as a mediator of IL-6 signaling, binding to IL-6 responsive agents in acute phase response genes such as TNF, IL-8 and G-CSF (Akira et al., Embo J., 1990, 9, 1897-1906; Descombes et al., Genes Dev., 1990, 4, 1541-1551) isolated from the liver and primarily regulates hormone responsiveness and oxidative stress responses (Descombes et al., Genes Dev., 1990, 4, 1541-1551). Studies of tissue distribution and developmental expression patterns showed that C/EBP beta is found the liver, lung, spleen, kidney, brain and testis with the highest expression found in the lung (Descombes et al., Genes Dev., 1990, 4, 1541-1551). Disclosed in U.S. Pats. 5,215,892 and 5,360,894 are the nucleic acid sequence of C/EBP beta as well as plasmids and host cells for the expression of the recombinant protein (Kishimoto et al., 1994; Kishimoto et al., 1993).
C/EBP-deficient mice have been generated for five of the six members of the C/EBP family and these have been characterized for system-specific phenotypic abnormalities. Mice lacking C/EBP beta demonstrate defective carbohydrate metabolism, immunodeficiency, defective Th1 response and female sterility. They also have a low level of expression of phosphoenolpyruvate carboxykinase (Park et al., J. Biol. Chem., 1999, 274, 211-217; Yamada et al., J. Biol. Chem., 1999, 274, 5880-5887) and demonstrate perinatal lethality suggesting involvement of C/EBP beta in gluconeogenic pathways (Arizmendi et al., J. Biol. Chem., 1999, 274, 13033-13040; Greenbaum et al., J. Clin. Invest., 1998, 102, 996-1007). These mice are highly susceptible to infection by a wide range of pathogens indicating a critical role for C/EBP beta in bactericidal responses (Tanaka et al., Cell, 1995, 80, 353-361).
In addition to stage-specific expression level variations, the C/EBP members also undergo multiple isoform expression arising from alternative start positions, for the alpha and beta isoforms, in 5' upstream open reading frames (Geballe and Morris, Trends Biochem. Sci., 1994, 19, 159-164; Lincoln et al., J. Biol. Chem., 1998, 273, 9552-9560). The steady-state level of the various pools of transcripts also changes as a function of age and stress challenges (Hsieh et al., Mol. Biol. Cell, 1998, 9, 1479-1494). In mice the expression of certain transcripts of one isoform has also been shown to regulate the expression of other C/EBP isoforms (Burgess-Beusse et al., Hepatology, 1999, 29, 597-601).
C/EBP beta occurs as two isoforms in the cell, a full-length 32-kDa form, known as LAP and a shorter form known as LIP. The dimerization of these two forms attenuates transcription of the C/EBP beta gene and therefore represents an autoregulatory mechanism of expression (Descombes et al., Genes Dev., 1990, 4, 1541-1551).
In disease states, C/EBP beta has been implicated in the development of diabetes and cancer. Studies comparing normal and tumorigenic tissue from human ovaries demonstrated a higher level of expression of C/EBP alpha and beta in the tumor tissues, irrespective of stage or grade of tumor (Sundfeldt et al., Br. J. Cancer, 1999, 79, 1240-1248). In the rat pancreas, it was shown that C/EBP beta expression is upregulated upon chronically elevated levels of glucose, possibly contributing to impaired insulin secretion in severe type II diabetes (Lu et al., J. Biol. Chem., 1997, 272, 28349-28359; Seufert et al., J. Clin. Invest., 1998, 101, 2528-2539).
The pharmacological modulation of C/EBP beta activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of C/EBP beta and to date, investigative strategies aimed at modulating C/EBP beta function have involved the use of antibodies and gene knock-outs in mice. However, these strategies are untested as therapeutic protocols and consequently there remains a long felt need for agents capable of effectively inhibiting C/EBP beta function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of C/EBP beta expression.
The present invention provides compositions and methods for modulating C/EBP beta expression, including modulation of both the long and short isoforms of C/EBP beta.